Bottom Line:
In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells.Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment.Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

ABSTRACTBacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

pone-0014682-g005: Internalized Bin is associated with its receptor but is not bound to vacuoles.(A, C and E) MDCK-Cpm1 cells were incubated with BinA labeled with Alexa 488 (BinA-Al488) and BinB labelled with Alexa 543 (BinB-Al543) at 37°C for 10 min (A), 30 min (C) or 6h (E) and processed for confocal microscopy analysis. At any time point, BinA-Al488 and BinB-Al543 were found colocalized in multiple intracellular vesicles. (B) Cells transfected with GPI-GFP and treated for 10 min with unlabelled BinA and BinB-Al543. Vesicles positive for BinB-Al543 were also stained by GPI-GFP. (D) Immunodetection of Cpm1 in MDCK-Cpm1 cells treated for 30 min with unlabelled BinA and BinB-Al543. Cpm1 signal and BinB-Al543 labeled vesicles were found colocalized. (E) BinA-Al488 and BinB-Al543-containing vesicles were found clustered between the vacuoles but not decorating the membrane of these vacuoles nor inside. Bars, 5 µm.

Mentions:
We next looked for Bin internalization in MDCK-Cpm1 cells by directly labelling BinA with Alexa 488 (Al488) and BinB with Alexa 543 (Al543). After 10 min of intoxication we could detect both BinA-Al488 and BinB-Al543 in small vesicles located mainly at the cell periphery (Fig. 5A). Then we proceeded to an intoxication with a combination of the BinB-Al543/BinA in cells transfected with a GPI-GFP construct. This construct consists of the decay accelerating factor (DAF) GPI anchor fused to GFP and serves as a valuable marker of compartments enriched in GPI-anchored proteins (GPI-APs) [29], [30]. At 10 minutes post-infection BinB-Al543 positive vesicles colocalized with the GPI-GFP marker suggesting that the toxin is internalized in association with its GPI-anchored receptor Cpm1 (Fig. 5B). After 30 min of intoxication, numerous endocytic vesicles containing both BinA-Al488 and BinB-Al543, as revealed by the colocalization of the two components, could be detected and were distributed throughout the cytoplasm (Fig. 5C). Immunodetection of the Bin receptor, the GPI-anchored protein Cpm1, revealed that it colocalized with the Bin endocytic vesicles (Fig. 5D). In vacuolated cells observed after 6 h of treatment, vesicles containing BinA-Al488 and BinB-Al543 were placed side by side with the vacuoles. Nevertheless the toxin was detected neither on the vacuole membranes nor inside the vacuoles (Fig. 5E, Fig.S4, video S7 and video S8). These results suggest that Bin is internalized in association with its receptor but is not targeted by the vacuolating autolysosomal compartment.

pone-0014682-g005: Internalized Bin is associated with its receptor but is not bound to vacuoles.(A, C and E) MDCK-Cpm1 cells were incubated with BinA labeled with Alexa 488 (BinA-Al488) and BinB labelled with Alexa 543 (BinB-Al543) at 37°C for 10 min (A), 30 min (C) or 6h (E) and processed for confocal microscopy analysis. At any time point, BinA-Al488 and BinB-Al543 were found colocalized in multiple intracellular vesicles. (B) Cells transfected with GPI-GFP and treated for 10 min with unlabelled BinA and BinB-Al543. Vesicles positive for BinB-Al543 were also stained by GPI-GFP. (D) Immunodetection of Cpm1 in MDCK-Cpm1 cells treated for 30 min with unlabelled BinA and BinB-Al543. Cpm1 signal and BinB-Al543 labeled vesicles were found colocalized. (E) BinA-Al488 and BinB-Al543-containing vesicles were found clustered between the vacuoles but not decorating the membrane of these vacuoles nor inside. Bars, 5 µm.

Mentions:
We next looked for Bin internalization in MDCK-Cpm1 cells by directly labelling BinA with Alexa 488 (Al488) and BinB with Alexa 543 (Al543). After 10 min of intoxication we could detect both BinA-Al488 and BinB-Al543 in small vesicles located mainly at the cell periphery (Fig. 5A). Then we proceeded to an intoxication with a combination of the BinB-Al543/BinA in cells transfected with a GPI-GFP construct. This construct consists of the decay accelerating factor (DAF) GPI anchor fused to GFP and serves as a valuable marker of compartments enriched in GPI-anchored proteins (GPI-APs) [29], [30]. At 10 minutes post-infection BinB-Al543 positive vesicles colocalized with the GPI-GFP marker suggesting that the toxin is internalized in association with its GPI-anchored receptor Cpm1 (Fig. 5B). After 30 min of intoxication, numerous endocytic vesicles containing both BinA-Al488 and BinB-Al543, as revealed by the colocalization of the two components, could be detected and were distributed throughout the cytoplasm (Fig. 5C). Immunodetection of the Bin receptor, the GPI-anchored protein Cpm1, revealed that it colocalized with the Bin endocytic vesicles (Fig. 5D). In vacuolated cells observed after 6 h of treatment, vesicles containing BinA-Al488 and BinB-Al543 were placed side by side with the vacuoles. Nevertheless the toxin was detected neither on the vacuole membranes nor inside the vacuoles (Fig. 5E, Fig.S4, video S7 and video S8). These results suggest that Bin is internalized in association with its receptor but is not targeted by the vacuolating autolysosomal compartment.

Bottom Line:
In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells.Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment.Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.

ABSTRACTBacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.